A radioactive antigen-binding assay for the measurement oof antibody to Haemophilus influenzae type b capsular polysaccharide

Kuo, Jen-Tsai; Monji, N.; Schwalbe, Richard; McCoy, Donald W.

doi:10.1016/0022-1759(81)90034-x

Cited by 50 publications

(20 citation statements)

References 19 publications

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“…Serology. Except where noted, the concentrations of total anti-Hib PS antibody were measured by a RABA previously described (21,22). This assay uses 50 pL of test sample diluted to a final vol of 500 pL, and polyethylene glycol radioimmune precipitation.…”

Section: Methodsmentioning

confidence: 99%

“…In brief, a RABA was performed using 0.075 ng/mL of the radiolabeled Hib PS antigen instead of the usual concentration of 3.0 ng/mL to insure that the antibody concentration was in excess. Also, to conform to the Griswold procedure, the RABA was modified to use a final vol of 70 pL instead of 500 pL (21), and an equal vol of saturated ammonium sulfate was used to precipitate antibody and antigen-antibody complexes instead of polyethylene glycol radioimmune precipitation (21). The fraction of bound antigen was calculated from counting the radioactivity of the pellets at different antibody dilutions.…”

Section: Methodsmentioning

confidence: 99%

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Variability in the Functional Activity of Vaccine-Induced Antibody to Haemophilus influenzae Type b

1990

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ABSTRACT. Sera from 3 of 30 adults vaccinated with Haemophilus influenzae type b polysaccharide vaccine (Hib PS) had poor complement-mediated bactericidal activity despite the presence of anti-Hib PS antibody concentrations of 8.6 to 20.5 ~g / m L .These "nonkiller" antibodies killed ~0 . 4 log cfu/mL compared to >3 logs with all but one of the other sera. To investigate the basis of this poor functional activity, we characterized in detail the IgG antibodies to Hib PS present in two of the nonkiller sera, and compared the results with two of the "killer" sera. The latter were selected based on comparable levels of total antibody to Hib PS. No consistent differences were found between the relative proportions of IgG or IgA antibody to total anti-Hib PS antibody, or the respective ratios of IgGl to IgG2 antibody in the nonkiller and killer sera. IgG fractions, and IgG affinity purified antibody to Hib PS were prepared. When tested at 2 pg/mL of antibody, the IgG fractions from the two nonkiller sera had much lower bactericidal activity than the corresponding fractions from the killer sera (3 logs less killing), and the former also had lower complement-mediated opsonic activity (20 and 13% uptake by human PMN compared to 62 and 93%). These data show the striking variability in the functional activity of vaccine-induced antibody to Hib PS. Antibody functional activity is likely to be affected by a number of factors but one important variable appears to be avidity since the IgG anti-Hib PS antibody from the two nonkiller sera had 2-to 5-fold lower avidity than the IgG antibody from the two killer sera. Therefore, measurement of antibody avidity in studies of Hib vaccine immunogenicity would be informative

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Variability in the Functional Activity of Vaccine-Induced Antibody to Haemophilus influenzae Type b

1990

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“…Protein was determined by the method of Lowry as modified by Peterson (11) employing bovine serum albumin (BSA) as the standard. Amino acid analysis was performed on a Durrum D-500 analyzer ( Anticapsular antibody in these sera was determined by radioimmune assay using 3H-labeled Hib capsule as described by Kuo and co-workers (12). Anti-LPS and anti-P2 antibodies were quantitated by enzyme-linked immunosorbent assay (ELISA) (13).…”

Section: Methodsmentioning

confidence: 99%

“…LPS-Sepharose preparation and antibody absorptions were performed as previously described (13). For preparation of PRP-Sepharose, PRP was purified from the supernatant of stationary phase broth cultures by sequential precipitation with ethanol, Cetavlon, and ethanol, as described by Kuo and co-workers (12). The final PRP precipitate was dissolved in 20 mM sodium phosphate, pH 6.9, and residual contaminants were removed by absorption with hydroxylapatite.…”

Section: Methodsmentioning

confidence: 99%

Purification and comparison of outer membrane protein P2 from Haemophilus influenzae type b isolates.

Munson¹,

Shenep²,

Barenkamp³

et al. 1983

J. Clin. Invest.

133

146

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A B S T R A C T Haemophilus influenzae type b isolateshave been subdivided based on differences in major outer membrane protein (OMP) profiles resolved on gradient and modified Laemmli sodium dodecyl sulfate-polyacrylamide gel electrophoresis systems. Although 21 subtypes have been observed, 86% of invasive isolates have one of five common subtypes and 71% of isolates have one of three subtypes. In isolates with two of the most common outer membrane subtypes, one major OMP has an apparent molecular weight of 37,000. In isolates with another common OMP subtype, a cross-reactive protein with an apparent molecular weight of 36,500 is observed. All three proteins have been designated P2. Protein P2 from these prototype isolates was solubilized with Zwittergent 3-14 and purified to homogeneity. Based on amino acid compositions, cyanogen bromide cleavage products, staphylococcal V8 protease, and chymotryptic peptide maps, the P2 protein from the three isolates has been highly conserved. Rabbit antibody prepared against P2 from one strain was cross-reactive with P2 isolated from the other two heterologous strains by Western blot. This antibody passively protected infant rats against type b Haemophilus infection caused by the homologous organism, but not against challenge by a strain with the heterologous 36,500 mol wt P2 protein. Thus, although the P2 protein from isolates with different OMP subtypes are closely related, the protection experiments suggest that determinants on This work was presented in part at the Annual Meeting of the

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“…Total anti-PRP antibodies were assayed in all subjects using a radio-labelled antigen binding assay [13] with a cut-off of 0.15 g/ ml. In a random subset of 46 subjects IgG subclasses were measured using an enzyme linked immuno-sorbent assay (ELISA) method developed at SmithKline Beecham Biologicals.…”

Section: Reactogenicitymentioning

confidence: 99%

Evidence for induction of polysaccharide specific B-cell-memory in the 1st year of life: plain Haemophilus influenzae type b - PRP (Hib) boosters children primed with a tetanus-conjugate Hib-DTPa-HBV combined vaccine

Zepp¹,

Schmitt²,

Kaufhold³

et al. 1996

Eur J Pediatr

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The tetanus-PRP conjugate vaccine not only elicited a good primary humoral response, but also induced immunological memory so that the infants were able to mount a large and rapid immune response to subsequent exposure to plain PRP, indicating that protection against circulating wild-type Hib had been generated. Successful induction of immunological memory occurred even when there was no measurable humoral anti-PRP response to the primary course. Tetanus-PRP conjugate vaccine can be used in combination with DTPa-HBV vaccine, when administered separately or as a single injection in the same syringe, in primary immunisation schedules at 3, 4 and 5 months of age.

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A radioactive antigen-binding assay for the measurement oof antibody to Haemophilus influenzae type b capsular polysaccharide

Cited by 50 publications

References 19 publications

Variability in the Functional Activity of Vaccine-Induced Antibody to Haemophilus influenzae Type b

Variability in the Functional Activity of Vaccine-Induced Antibody to Haemophilus influenzae Type b

Purification and comparison of outer membrane protein P2 from Haemophilus influenzae type b isolates.

Evidence for induction of polysaccharide specific B-cell-memory in the 1st year of life: plain Haemophilus influenzae type b - PRP (Hib) boosters children primed with a tetanus-conjugate Hib-DTPa-HBV combined vaccine

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